Transfer Of Interleukin-4and Interleukin-10in Experimental Murine Colitis

Background&objectivesInflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammation involving genetic factors, environmental triggers and immunological factors. IBD, including Crohn’s disease and ulcerative colitis, are chronic inflammatory and frequently relapsing disease. The incidence of IBD is higher in USA and European countries, and it has been recently increased in China. Although the pathogenesis of IBD is still not completely understood, there is increasing evidence suggest that an imbalance of Thl/Th2immune response plays an important role in the pathogenesis of IBD. CD4+T cell is divided into Thl cells and Th2cells according the secreted cytokines and mediated immune function. Thl cells predominantly secrete interferon-y (IFN-y),interleukin-2(IL-2) and tumor necrosis factor-a (TNF-α),while Th2cells secrete interleukin-4,5and10(IL-4,IL-5and IL-10).Cytokines plays a core role in the pathogenesis of IBD. There are two types of cytokines, pro-inflammatory cytokines and anti-inflammatory cytokines. Disturbed balance of pro-inflammatory cytokines and anti-inflammatory cytokines is crucial in the initiation and progression of IBD. Pro-inflammatory cytokines such as IL-1, IL-6, TNF-α and IFN-γ are produced by monocytes and macrophages and are involved in cell-mediated immune responses. Anti-inflammatory cytokines such as IL-4, IL-5, IL-10are produced by T cells and are involved in humoral immune response. Of these, IL-4and IL-10are two of the most vital anti-inflammatory cytokines.IL-10is produced by various cells, including T cells (CD4+and CD8+cells), B cells, monocytes, macrophages and dendritic cells. It is coded by five exons on Chromosome1. It inhibits the production of TNF-a, INF-y and IL-6and the activation of inflammatory cells as a major negative regulatory factor. It is widely accepted that IL-10can prevent or cure IBD. IL-10-deficient mice spontaneously develop intestinal inflammation similar to CD. Also, IL-10treatment can suppress or alleviate the development of IBD in animal models.Human IL-4is about20KD glycoprotein and composed of129amino acids. The coding gene locates in Chromosome5, which consists of four exons and three introns. It is the longest lymphoid factor, approximately10Kb in length. Murine IL-4is mainly produced by lymphocyte, inhibiting the production of IL-1, IL-6, IL-8and TNF-a while inducing the production of IL-1α. It is necessary to maintain intestinal immunity. IL-4can regulate the balance of Th1/Th2cells and suppress Thl type inflammatory disease. Recent evidence demonstrate that the patients with UC have a lower level of cells secreted IL-4and a reduced IL-4mRNA expression. In contrast, some studies show that IL-4also can promote Thl type response by enhancing the production of IFN-y and TNF-α. In brief, IL-4acts as a complicated role in cell-mediated immune with an unclear mechanism.Gene therapy could be adopted by targeted cells with various factors or cytokines to upregulate their expression. Alternatively, genetic manipulation can be achieved in local mucosal by means of plasmid vectors in vivo, such as plasmid, liposome, retroviruses, and adenovirus. Restructured AAV (rAAV) containing no virus gene has been broadly used because of their high transfection efficiency. Although viral vectors have been used successfully in preclinical studies, the limitation size of inserted gene and the potential for toxicity of viruses, especially upon chronic administration, is a major concern when it comes to clinical reality.There are three primary animal models of IBD, including gene transfer ones, immunological ones and chemical ones. The model induced by2,4,6-trinitrobenzene sulfonic acid (TNBS) has been widely used. Colitis can be induced in susceptible strains of mice by instillation of the haptenating substance TNBS in ethanol. Ethanol is required to break the mucosal barrier, whereas TNBS is believed to haptenize colonic autologous or microbiota proteins rendering them immunogenic to the host immune system. TNBS model is similar to human CD by triggering Thl type immune response. Although the lack of acute stage performance and high mortality, the administration is simple, economic and easily repeatable. It is a classic model and available for new drugs selection and mechanism research.At present, conventional treatment for IBD is anti-inflammatory drugs, such as5-ASA, glucorticoids and immunosuppresssants. However, these drugs often trigger undesirable adverse effects and fail to bring satisfactory results. With the development of investigation in the etiology of IBD, an interest in new therapeutic options, that is biologic therapy, has around recently. TNF-a, as a crucial pro-inflammatory cytokine of IBD, exacerbates mucosal inflammation and is likely to be a focal point of the inflammatory cascade in IBD. The most effective anti-TNF-a agent is Infliximab, also as the only one approved by the US Food and Drug Administration(FDA) to treat patients with untreatable CD. Nevertheless, it is often accompanied with undesirable and potentially serious side effects, including infusion reaction, delayed hypertension, congestive heart failure and opportunistic infection, etc. Moreover, it has little effect on patients with UC.As the background showed above, we investigated the therapeutic effects of liposome-mediated gene transfer of IL-4and IL-10in IBD animal models. The protocol was:1. Constructing eukaryotic expression vector pcDNA3.0-mIL-4and pcDNA3.0-mIL-10.2. Detecting the expression of recombinant plasmids in vitro by ELISA and Western-blotting methods.3. Inducing mice colitis models by TNBS.4. Transfection of pcDNA3.0-mIL-4, pcDNA3.0-mIL-10or combinations of pcDNA3.0-mIL-4and pcDNA3.0-mIL-10in mice by means of liposome package.5. Detecting the changes of IFN-y expression, TNF-α and IL-6mRNA expressed in IBD mice.Methods1. mIL-4and mIL-10were amplified from BALB/c mouse spleen mRNA by RT-PCR, and ligated orientatively into the eukaryotic expressing plasmid pcDNA3.0to construct the recombinant plasmids.2. Recombinant plasmids were identified with EILISA or Western-blotting methods by transfected293T cells.3.Preparation of IBD by TNBS in mice:A total of70male BALB/c mice were divided into vehicle group and6experimental groups, pcDNA3.0-mIL-4group(Liposome/IL-4), pcDNA3.0-mIL-10group(Liposome/IL-10), pcDNA3.0-mIL-4and pcDNA3.0-mIL-10group(Liposome/IL-4+IL-10), TNBS group, pcDNA3.0group, Liposome group (10mice per group).100ul TNBS-ethanol solutions was administered rectally to each mouse in model groups and100ul50%ethanol was administered rectally to each mouse in vehicle. Mice received a peritoneal injection of plasmid/Liposome complex, Liposome or saline24h after TNBS administration. Mice were all executed on day7.4. The distal colon were fixed in4%formaldehyde, embedded in paraffin, sectioned at5um, and stained with haematoxylin and eosin (HE). Histological assessment was performed by two experienced pathologist blinded to treatment allocation.5. Detecting IFN-y expressed in colon mucosa of mice by ELISA.6. Detecting TNF-a, IL-6mRNA expressed in colon mucosa of mice by quantitative RT-PCR.7. Data analyses:All the data were analyzed by SPSS13.0software. Data are expressed as mean±SD if they obeyed normality distribution and expressed as median if they did not obey normality distribution. Statistical significance was tested using One-way ANOVA (LSD or Dunnett’s T3methods between groups), K Independent-Samples Nonparametric Test and ANOVA for Repeated Measures. Statistical significance was set at P values less than0.05.Results1. Two recombinant plasmids, pcDNA3.0-mIL-4and pcDNA3.0-mIL-10, were successfully constructed and identified by restriction enzymes digestion and sequencing.2. mIL-4expression was evaluated by ELISA method, mIL-10expression was detected by Western blotting method.3. The recombinant plasmids capsuled by liposome can be expressed in vivo by means of intraperitoneal injection.4. Disease activity index (DAI) scores of mice in Liposome/IL-4and Liposome/IL-10treated group were lower than that in TNBS group; mice in Liposome/IL-4+IL-10treated group obtained the same scores as those in TNBS group.5. The body weight of each group except the vehicle group decreased mostly on day2and day3. When compared with the constant reduction of body weight in the TNBS group and Liposome/IL-4+IL-10treated group, mice in Liposome/IL-4and Liposome/IL-10treated group had significant recovery of body weight from day3.6. Histological scores of colon in Liposome/IL-4and Liposome/IL-10treated group were obviously lower than those of TNBS group and Liposome/IL-4+IL-10 treated group (x2=37.118, P<0.001).7. IFN-y expressed in colon mucosa of mice in Liposome/TL-4, Liposome/IL-10and Liposome/IL-4+IL-10treated group were obviously lower than TNBS group. But the level of IFN-y in Liposome/IL-4+IL-10treated group is significantly higher than that in Liposome/IL-4or Liposome/IL-10treated group (x2=34.148, P<0.001).8. TNF-a and IL-6mRNA expressed in colon mucosa of mice in Liposome/IL-4, Liposome/IL-10and Liposome/IL-4+IL-10treated group were lower than those of TNBS group (P<0.05)Conclusions1. The expression of recombinant plasmids of pcDNA3.0-mIL-4and pcDNA3.0-mIL-10can be both detected in vivo or in vitro.2. IL-4and IL-10gene transfer can reduce DAI scores and alleviate intestinal inflammation. Combined administration of plasmids encoding IL-4and IL-10has little effect on colitis development.3. IL-4and IL-10gene transfer result in downregulation of pro-inflammatory cytokines expression, IFN-y, TNF-a and IL-6. Combined administration of plasmids encoding IL-4and IL-10has a weak effect on that than any single gene transfer.4. IL-4and IL-10gene transfer are effective in the treatment of IBD, however, the cure effect of combined administration of plasmids encoding IL-4and IL-10is not expectedly obvious.